Optically trapping confocal Raman microscopy of individual lipid vesicles: Kinetics of phospholipase A(2)-catalyzed hydrolysis of phospholipids in the membrane bilayer
ABSTRACT Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.
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ABSTRACT: Apoptosis is a tightly controlled process in mammalian cells. It is important for embryogenesis, tissue homoeostasis, and cancer treatment. Apoptosis not only induces cell death, but also leads to the release of signals that promote rapid proliferation of surrounding cells through the Phoenix Rising (PR) pathway. To quantitatively understand the kinetics of interactions of different molecules in this pathway, we developed a mathematical model to simulate the effects of various changes in the PR pathway on the secretion of prostaglandin E2 (PGE2), a key factor for promoting cell proliferation. These changes include activation of caspase 3 (C3), caspase 7 (C7), and nuclear factor κB (NFκB). In addition, we simulated the effects of cyclooxygenase-2 (COX2) inhibition and C3 knockout on the level of secreted PGE2. The model predictions on PGE2 in MEF and 4T1 cells at 48 hours after 10-Gray radiation were quantitatively consistent with the experimental data in the literature. Compared to C7, the model predicted that C3 activation was more critical for PGE2 production. The model also predicted that PGE2 production could be significantly reduced when COX2 expression was blocked via either NFκB inactivation or treatment of cells with exogenous COX2 inhibitors, which led to a decrease in the rate of conversion from arachidonic acid to prostaglandin H2 in the PR pathway. In conclusion, the mathematical model developed in this study yielded new insights into the process of tissue regrowth stimulated by signals from apoptotic cells. In future studies, the model can be used for experimental data analysis and assisting development of novel strategies/drugs for improving cancer treatment or normal tissue regeneration.PLoS Computational Biology 02/2014; 10(2):e1003461. DOI:10.1371/journal.pcbi.1003461 · 4.83 Impact Factor
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ABSTRACT: We present the first experimental study of self-forming synthetic lipids, trademarked as QuSomesTM, using Raman spectroscopy in the spectral range of 500 to 3100 cm-1. Raman spectra of these new artificial lipids composed of 1,2- dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol (GDO-12) have been obtained in pure form and in aqueous suspensions with Phosphate Buffered Saline (PBS) by using an inverted confocal laser-tweezers-Raman-microscopy system. This spectrometer works with an 80 mW diode-pumped solid-state laser, operating at a wavelength of 785 nm in the TEM00 mode. The laser is used both for optical trapping and Raman excitation. The two amphiphiles considered in this study, differ in their hydrophobic chain length and contain similar units of hydrophilic polyethylene glycol (PEG) head groups. Such synthetic PEG coated lipids exist in liquid form at room temperature and spontaneously form liposomes (nano type vesicles) upon hydration. In this work, we have focused on the band assignments for the spectra of single QuSomesTM nano particles in pure form and in aqueous media acquired by means of Raman spectroscopy. In particular, we have found that the most prominent peaks in the studied spectral region are dominated by vibrational modes arising from C-C and C-H bonds. Furthermore, we have noticed that some of the distinct peaks observed below 1800 cm-1 in pure sample are preserved in aqueous environment. These retained intense bands are located at 1449, 1128, 1079, and 1065 cm-1. This effect might be due to the strong chain-chain interactions, because the chains have to orient themselves and become tightly packed in the vesicles wall rather than adopt random orientations in bulk. This technique has proven to be an excellent tool to establish the fingerprint region revealing the molecular structure and conformation of QuSomesTM particles. The Raman spectroscopic data of these novel lipids and its vesicles formed in suspensions confirm high stability and are therefore considered as potential candidate for varieties of future applications including lipid based novel substances and drug delivery systems.Proceedings of SPIE - The International Society for Optical Engineering 01/2008; DOI:10.1117/12.804822 · 0.20 Impact Factor
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ABSTRACT: Vibrational spectroscopy has been used to elucidate the temperature dependence of structural and conformational changes in lipids and liposomes. In this work, the thermal properties of lipid-based nanovesicles originating from a newly developed self-forming synthetic PEGylated lipids has been investigated by variable-temperature Fourier-transform infrared (FTIR) absorption and Raman spectroscopic methods. Thermally-induced changes in infrared and Raman spectra of these artificial lipid based nanovesicles composed of 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) were acquired by using a thin layered FTIR spectrometer in conjunction with a unique custom built temperature-controlled demountable liquid cell and variable-temperature controlled Raman microscope, respectively. The lipids under consideration have long hydrophobic acyl chains and contain various units of hydrophilic polyethylene glycol headgroups. In contrast to conventional phospholipids, this new kind of lipid is forming liposomes or nanovesicles spontaneously upon hydration, without supplying external activation energy. We have found that the thermal stability of such PEGylated lipids and nanovesicles differs greatly depending upon the acyl chain-lengths as well as associated head group units. However, the thermal behavior observed from both spectroscopic vibrational techniques are in good agreement.Proceedings of SPIE - The International Society for Optical Engineering 02/2011; DOI:10.1117/12.875853 · 0.20 Impact Factor